BCMB 3100 Lipids (Text Chapters 11, 12, 13) Definition Major classes Fatty acids Triacylglycerol Glycerophospholipids Sphingolipids Cholesterol : water insoluble organic compounds in living organisms Lipids are hydrophobic or amphipathic In BCMB3100 we will emphasize *phospholipids *glycolipids *cholesterol (steroid) : main lipids in most biological membranes : 2nd most abundant lipid in membranes (abundant in CNS) from animals and plants 1
Structural relationships of major lipid classes Structure and nomenclature of fatty acids 2
See Table 11.1 You must be able to draw the structure of those marked * * * * * * common name IUPAC name 18:0 stearate Octadecanoate 18:1 oleate cis- 9-Octadecenoate 18:2 linoleate cis,cis- 9,12-Octadecadienoate Unsaturated FA - at least one C-C double bond Saturated FA - no C-C double bonds 3
Fig. 11.1 Page 181/191 Pg. 182/192 4
Structural relationships of major lipid classes Structure of a triacylglycerol (a) Glycerol backbone (b)triacylglycerol (a) are a neutral storage form of fatty acids (b) See Pg. 183/193 5
An EXAMPLE A triglyceride molecule. Left: glycerol; right (top to bottom): palmitic acid, oleic acid, alpha-linolenic acid Note: the types of fatty acyl groups present in any given triacylglycerol may vary. MEMBRANE LIPIDS: 3 major types = phospholipids, glycolipids, & cholesterol : most abundant class of lipids in membranes (note: triacylglycerols most abundant on mass basis in mammals); derived from glycerol or sphingosine *lipids from glycerol = phosphoglycerides (also called glycerophospholipids) * phosphoglycerides consist of glycerol backbone, two fatty acids & a phosphorylated alcohol See Fig. 11.5 Fatty acid Fatty acid G L Y C E R O L Phosphate alcohol 6
Fatty acids in biological organisms Fatty acid chains (long aliphatic tails) in phospholipids & glycolipids contain even # of carbons (12-20) with 16 and 18 being most common Fatty acids can be or Under physiological conditions fatty acids are ionized (pka 4.5-5.0) Fig 9.1 Structural relationships of major lipid classes 7
(a) Glycerol 3-P and (b) phosphatidate See Fig. 11.6 See Fig. 11.7 8
Phospholipases hydrolyze phospholipids : enzymes that catalyze hydrolysis of triacylglycerols : catalyze hydrolysis of glycerophospholipids Structural relationships of major lipid classes 9
(a) : structural backbone of sphingolipids (b) : sphingosine + fatty acid at C2 See Fig. 11.8 Ceramide See Fig. 11.8 (c) Sphingomyelin: present in plasma membrane & myelin sheath around neurons 10
See Pg. 186/196 Example of a Cerebroside: abundant in nerves Sugar-Sphingosine Fatty acid Structure of a galactocerebroside Example of a Ganglioside Ganglioside G M2 (NeuNAc in blue) Cell surface, cell-cell interactions (e.g. blood group antigens) Hexosaminidase A cleaves here Mutation Tay-Sachs disease 11
Structural relationships of major lipid classes Structure of the steroid cholesterol. Steroids are polyprenyl compounds See Pg. 187/197 Synthesized from isoprene In eukaryotes but NOT in most prokaryotes Other steroids: steroid hormones (estrogen estradiol, testosterone, corticosteriods), bile salts, sterols in plants, yeast, fungi 12
Cholesterol modulates the fluidity of mammalian cell membranes It is also a precursor of the steroid hormones and bile salts Stereo view of cholesterol Polar OH (red), fused ring system nearly planar 13
Waxes: esters of long-chain monohydroxylic alcohols and long-chain fatty acids (nonpolar) Waxes are very water insoluble and high melting point They are widely distributed in nature as protective waterproof coatings on leaves, fruits, animal skin, fur, feathers and exoskeletons Myricyl palmitate, a wax Palmitate portion Myricyl alcohol portion Eicosanoids: oxygenated derivatives of C20 polyunsaturated fatty acids (e.g. arachidonic acid) can cause constriction of blood vessels involved in blood clot formation mediator of smooth-muscle contraction and bronchial constriction seen in asthmatics Arachidonic acid and three eicosanoids 14
Some vitamins are Lipid Vitamins Four lipid vitamins: A, D, E, K (All contain rings and long, aliphatic side chains All are highly hydrophobic The lipid vitamins differ widely in their functions * Examples of isoprenoids * * * * See Fig. 15.18 / 15.19 for structures Fig. 15.18/15.19 15
BCMB 3100 - Lipids Biological Membranes Micelles Lipid Bilayer Peripheral membrane proteins Integral membrane proteins Lipid-anchored Transport across membranes Signal Transduction Structure of a typical eukaryotic plasma membrane See Fig. 12.1; 12.8 16
Highly selective permeability barriers that surround cells & cellular compartments Sheetlike structures of ~60-100Å Consists mostly of lipids & proteins in ratio of 1:4 to 4:1 (typical 40% lipid; 60% protein). Lipids & proteins may be glycosylated. Lipids in biological membranes are : hydrophilic (polar) head group & hydrophobic tail. Spontaneously form bilayers in aqueous solution. Fig. 12.1 17
Membrane lipid and bilayer Lipids in biological membranes include phospholipids, glycosphingolipids, cholesterol (in some eukaryotes) Stereo view of cholesterol Polar OH (red), fused ring system nearly planar 18
Amphipathic nature of of cerebroside 19
Amphipathic lipids can take two different forms in aqueous media: or : a globular structure in which polar head groups are on the surface and hydrocarbon tails are on the inside Salts of fatty acids tend to form micelles. Micelles usually are < 200 m in diameter. Structure and nomenclature of fatty acids 20
: favored structure for phospholipids & glycolipids since lipids with two fatty acyl chains are too large to fit into the center of a micelle. Bilayers can have large dimensions (10 7 Å, 1mm) (recall diameter = ~60-100Å) Lipid bilayers self-assemble due to hydrophobic interactions between hydrocarbon tails (main force), van der Waals attractive forces between hydrocarabon tails, & elecrostatic & H-bonding forces between polar head groups and water Biolayers are extensive, closed, and self-sealing A liposome Fig. 12.2 21
Preparation of liposomes Fig. 12.3 Lipid bilayers are permeability barriers to ions & polar molecules Lipid vesicles (liposomes): aqueous compartments enclosed by lipid bilayers. Small vesicles (~ 500 Å), large vesicles (~10 4 Å, 1 µm) A A A A Use lipid vesicles to measure membrane permeability 1. Form vesicles in solution containing A 2. Separate vesicles from free A 3. Measure flux of A out of vesicles Fig. 12.2 22
Results Permeability coefficient (cm/s) Na + 10-12 ; Trp 10-7 ; indole ~5x10-4 ; water ~5x10-3 Water & hydrophobic molecules readily traverse membranes while ions & most polar molecules do not Fig. 12.4 Lipid Bilayers and Membranes Are Dynamic Structures (a) Lateral diffusion is very rapid (b) Transverse diffusion (flip-flop) is very slow See Fig. 12.15 23
Experiment showing that lateral diffusion occurs in biological membranes via use of heterokaryons (Frye & Edidin) Diffusion of membrane proteins Fluorescence recovery after photobleaching (FRAP) evidence for fluid membrane. Fig. 12.14 24
Biological Membranes (cont.) Contain both embedded in the bilayer & on its surface. may function as pumps, gates, receptors, energy transducers & enzymes. Held together by noncovalent interactions Asymmetric: the two surfaces (faces) differ in properties Two dimensional fluids - lipids & proteins rapidly diffuse in plane of membrane but NOT across membrane - membrane proteins and lipids can rapidly diffuse laterally or rotate within the bilayer (Singer & Nicolson, 1972) Compositions of biological membranes vary considerably among species and cell types Freeze-fracture electron microscopy, shows the distribution of membrane proteins 25
Phase transition of a lipid bilayer Fluid properties of bilayers depend upon the flexibility of their fatty acid chains Ordered state: a rigid state in which all C-C bonds have trans conformation (all trans) Fluid state: a relatively disordered state in which some of the C-C bonds are in the gauche conformation Phase transition of a lipid bilayer Fig 12.5 26
Transition from rigid to partly fluid state occurs at T M, the T M depends on of fatty acyl chains & on degree of Rigid state favored by saturated fatty acyl chains Disordered state favored by cis double bound(s) (i.e. T M is lowered) Prokaryotes regulate membrane fluidity by varying # of double bonds & length of fatty acyl chains. As temperature changes from 42ºC to 27ºC ratio of saturated:unsaturated changes from 1.6 to 1 Packing of fatty acid chains in membrane is disrupted by double bounds and lowers Tm. Fig. 12.6 27
Effect of cholesterol on phase transition (T M ) of membranes In eukaryotes membrane fluidity is largely regulated by. Cholesterol moderates the fluidity of membranes (prevents tight packing of fatty acyl chains & blocks large motions Addition of 20 mol% cholesterol broadens phase transition Pure phospholipid bilayer has a sharp phase transition Fig 12.7 28
Cholesterol modulates fluidity of the membranes. Also, association with sphingolipids leads to cholesterol-rich regions called lipid rafts that may effect specific membrane-protein function. Lipid raft http://en.wikipedia.org/wiki/lipid_raft Structure of a typical eukaryotic plasma membrane 29
Three types membrane associated proteins : loosely bound to membrane by H-bonds or electrostatic forces, generally water soluble once released from membrane using high salt or ph. Often bound to integral membrane proteins : proteins firmly bound to membrane by hydrophobic interactions. Solubilized with detergents. Most have one or more membrane spanning domains (e.g. -helix with ~20 amino acids). Integral and peripheral membrane proteins Fig. 12.8 30
Stereo view of bacteriorhodopsin: an integral membrane protein See Fig. 12.9 Bacterial porin Fig. 12.10 Lipid-anchored membrane proteins: proteins covalently linked to lipid membrane Types of links: *direct amide or ester bond between amino acid and fatty acyl group such as myristate or palmitate *prenylation: link to an isoprenoid chain (e.g. farnesyl or geranylgeranyl) via the S of a Cys near the C terminus of the protein *glycosylphosphatidylinositol anchor: C-terminal -carboxyl of protein-phosphoethanolamineglycan-phosphatidylinositol 31
Lipid-anchored membrane proteins Glycosylphosphatidylinositolanchored protein (GPIanchored protein) (continued) direct ester-linked protein Prenylated protein 32
Characteristics of membrane transport. Small uncharged molecules can diffuse across membranes. Other molecules require protein assisted movement ATP Light e - transport Membrane transport through a pore or channel Central passage through waterfilled pore allows specific molecules to transverse the membrane (e.g. porin in mitochondria outer membrane) There are many types of channels; ion channels can transport ions much fast than pumps Examples: voltage-gated channels, ligand-gated channels, transient receptor potential channels 33
Types of passive and active transport See Fig. 12.19/12.18 Active & passive transporters undergo a conformational change to drive transport 34
Active transporters move molecules against a concentration gradient: 1º transporters use 1º energy source (e.g. light, ATP, electron transport); 2º transporters driven by ion gradient Primary active transporter: Na + -K + ATPase Secondary active transporter: glucose transporter see Fig. 12.16/ 12.20/12.19 Active transport in E. coli Oxidation of S red generates a transmembrane proton gradient Movement of H + down its gradient drives lactose transport (lactose permease) 35
Potassium ion channel (1) Fig. 12.22 Potassium ion channel (2) Fig. 12.23 Selectivity filter of K+ ion channel 36
Potassium ion channel (3) Fig. 12.24 Energetic basis of ion selectivity in K+ ion channel Potassium ion channel (4) Fig. 12.24 Energetic basis of ion selectivity in K+ ion channel. 37
Potassium ion channel (5) Fig. 12.25 Rapid rate of K+ movement due to structure of channel and electrostatic repulsion of incoming K+ Molecules and complexes that are too large to be transported via transport proteins are transported in lipid vesicles out of the cell via exocytosis, and into the cell via endocytosis. We will not cover these processes in this course. 38
Signal transduction through a membrane Fig. 13.1 Three general classes of membrane receptor proteins: seven-transmembrane-helix receptors Dimeric receptors that recruit protein kinases Dimeric receptors that are protein kinases 78 39
General mechanism of signal transduction across a membrane (e.g. hormones) e.g. *G proteins Tyrosine kinase *Adenylate cyclase *Phospholipase C Common secondary messengers Fig. 13.2 40
G-protein cycle α: fatty acyl anchored gamma: prenyl-anchored G proteins are activated by binding to a receptor-ligand complex G-proteins are inactivated slowly by their own GTPase activity (kcat about 3/min) Understanding G Proteins: Hydrolysis of GTP to GDP and P i 41
Summary of the adenylyl cyclase signaling pathway Example of seventransmembrane-helix (7TM) receptor See Fig 13.6, 13.7; 13.8 Production, inactivation of camp Continued next slide 42
(continued) Activation of protein kinase A by camp See Fig. 13.7 43
Caffeine & theophylline inhibit camp phosphodiesterase Inhibition of camp phosphodiesterases prolongs the effects of camp This increases the intensity and duration of stimulatory hormones Inositolphospholipid signaling pathway See Fig. 13.11, 13.12 44
Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) produces IP 3 and diacylglycerol See Fig. 13.11 (continued) 45
Activation of receptor tyrosine kinases by ligandinduced dimerization See Fig. 13.15 (continued) Phosphorylated dimer phosphorylates cellular target proteins 46
(continued) Each domain catalyzes phosphorylation of its partner Insulin receptor and tyrosine kinase activity Insulin binds to 2 extracellular -chains Transmembrane -chains then autophosphorylate Tyrosine kinase domains then phosphorylate insulinreceptor substrates (IRSs) (which are proteins) 47
Insulin-stimulated formation of PIP 3 * See Figs. 13.17/13.17-13.21/13.22 *phosphatidyl-inositol 3,4,5-trisphosphate A different type of tyrosine kinase signal transduction: Growth hormone receptor for which binding brings together associated proteins with tyrosine kinase domains Fig. 13.13 48
Cross-phosphorylation of two JAK2 induced by hormone receptor dimerization Fig. 13.13 Fig. 13.14 Small G proteins (small GTPases) are a large superfamily of signalling proteins They include: Ras, Rho, Aft, Rab, and Ran Small GTPases cycle between an active GTP-bound form and an inactive GDP-bound form Small GTPases are smaller (20-25kd) and monomer compared to the larger (30-35 kd) and trimeric G proteins 49
Extra material Structure and nomenclature of fatty acids Saturated FA - no C-C double bonds Unsaturated FA - at least one C-C double bond Monounsaturated FA - only one C-C double bond Polyunsaturated FA - two or more C-C double bonds common name IUPAC name 18:0 stearate Octadecanoate 18:1 oleate cis- 9 -Octadecenoate 18:2 linoleate cis,cis- 9,12 -Octadecadienoate 50
Another method to measure membrane permeability Mueller & Rudin: dip paint brush into lipid membrane solution & paint across 1 mm diameter hole partitioned between two aqueous media. macroscopic bilayer membrane Measure electrical conductance from one media to the other 51